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and J. Herbert WaiteHua Zhao, Jason Sagert, Dong Soo Hwang

Perna viridisMussel Adhesive Protein fromGlycosylated Hydroxytryptophan in aGlycobiology and Extracellular Matrices:

doi: 10.1074/jbc.M109.022517 originally published online July 7, 20092009, 284:23344-23352.J. Biol. Chem.

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Glycosylated Hydroxytryptophan in a Mussel AdhesiveProtein from Perna viridis*SReceived forpublication,May 18,2009, andin revised form June 24,2009 Published, JBC Papers in Press,July 7, 2009, DOI 10.1074/jbc.M109.022517

Hua Zhao1, Jason Sagert1, Dong Soo Hwang, and J. Herbert Waite2

From the

Marine Sciences Institute, University of California at Santa Barbara, Santa Barbara, California 93106 and the

IndustrialBiotechnology Department, Institute of Chemical and Engineering Sciences, A*STAR, Singapore 627833

The 3,4-dihydroxyphenyl-L-alanine (Dopa)-containing pro-

teins of mussel byssus play a critical role in wet adhesion and

have inspired versatile new synthetic strategies for adhesives

and coatings. Apparently, however, not all mussel adhesive pro-

teins are beholden to Dopa chemistry. The cDNA-deduced

sequence of Pvfp-1, a highly aromatic and redox active byssal

coating protein in the green mussel Perna viridis, suggests that

Dopa may be replaced by a post-translational modification of

tryptophan. The N-terminal tryptophan-rich domain of Pvfp-1

contains 42 decapeptide repeats with the consensus sequencesATPKPW

1TAW

2K and APPPAW

1TAW

2K. A small collagen

domain (18 Gly-X-Y repeats) is also present. Tandemmass spec-

trometry of isolated tryptic decapeptides has detected both

C2-hexosylated tryptophan(W1)andC2-hexosylatedhydroxytryp-

tophan(W2),thelatterof whichisredox active. TheUV absorbance

spectrum of W2

is consistent with 7-hydroxytryptophan, which

represents an intriguing new theme for bioinspired opportunistic

wet adhesion.

The amino acid 3,4-dihydroxyphenyl-L-alanine (Dopa)3

occurs in many proteins of the mussel holdfast or byssus (1, 2)and has recently been incorporated into mussel-inspired syn-thetic polymers with versatile adhesive consequences (37).

One byssal protein in particular, mussel foot protein-1 (Mfp-1),has been investigated from over 15 mussel byssi, where it pro-tectively coats compliant collagen-like proteins in the threadcore (811). Mfp-1s typically contain 1015 mol % Dopa in ahighly conserved repeating peptide structure (8). In the blue

mussel Mytilus edulis fp-1 (Mefp-1), for example, the consen-sus decapeptide AKPSYPPTYK is repeated over 70 times intandem, and much of the tyrosine (Y) is converted to Dopa (Y*)(Fig. 1). In stark contrast to this, only trace levels of Dopa were

detected in Pvfp-1 from the green mussel Perna viridis (Lin-

naeus 1758) (12), a notoriously invasive fouling species origi-

nally from the Indo-Pacific region (13). Because P. viridis fp-1(Pvfp-1) and its homologue, Mefp-1, are both strongly aro-

matic, quinogenic, and composed of highly polar decapeptiderepeats (12), identifying the Dopa-mimetic substitutes inPvfp-1 has been a matter of considerable interest.

MATERIALS AND METHODS

Protein Isolation from Mussel FeetGreen mussels (P. viri-dis) were collected from Tampa harbor in Florida. The feet

were severed from about 100 freshly shucked mussels andstored at 80 C. Protein extraction from mussel feet wasadapted from a previous report (12). Frozen mussel feet (in lotsof 10 g of wet weight) were thawed and depigmented by scrap-ing with a scalpel. The depigmented feet were homogenized in

a glass tissue grinder with 50 ml of 5% acetic acid and twoprotease inhibitors (pepstatin A and leupeptin, both 30 mM).The homogenate was centrifuged (15,000 g, 4 C, 30 min),and the recovered supernatant (S1) was chilled in an ice bath.70% perchloric acid was added dropwise with stirring to a final

perchloric acid concentration of 1.4% (v/v). The mixture wasfurther stirred and centrifuged (15,000 g, 4 C) for 30 min.The supernatant (S2) was collected, dialyzed in 500 volumes of5% aceticacidfor 12h at4 C, freeze-dried at80 C,and resus-pended in 0.2 ml of 5% acetic acid.

P. viridis foot protein-1 (Pvfp-1) was purified in three stages:gel filtration, reversed phase HPLC, and finally, again, gel filtra-tion. Initial isolation was achieved by gel filtration chromatog-raphy on a Shodex KW-803 column (5 m, 8 300 mm). The

column wasequilibrated andelutedwith 5% aceticacid at a flowrate of 0.2 ml/min. A maximum volume of 200 l of S2 wasloaded onto the Shodex column/run. The eluted volume wasmonitored at 280 nm, and the fractions under the Pvfp-1 peakwere pooled (11.5 ml) and further resolved by C8 HPLC

(Brownlee Aquapore RP-300,7m,4.6250mm)ataflowrateof

1 ml/min with an acetonitrile gradient described by Ref. 12. Thecollected fractions wereassayed by redox cyclingafter acidurea gelelectrophoresis(12) andamino acid analysis as describedbelow toidentify Pvfp-1 containing fractions. Pvfp-1 fractions were pooled

and lyophilized at80 C.Afterresuspending in 200lof5%ace-tic acid, Pvfp-1-positive fractions were run once more on ShodexKW-803 (above conditions). Fractions eluting between 33 and 43min were redox active and had amino acid compositions consist-ent with previous studies (12). Purified Pvfp-1 fractions were

recovered by freeze-drying. The N terminusof purifiedPvfp-1 wassequenced by automated Edman degradation on a microse-quencer (model 2090; Porton Instruments, Tarzana, CA) with amodified gradient program (14).

*This work was supported,in whole or in part, by National Institutes of HealthGrant R01 DE018468. This work was also supported by National ScienceFoundation Grant MRSEC DMR05-20415.

S The on-line version of this article (available at http://www.jbc.org)containssupplemental Table S1 and Figs. S1S9.

This study is dedicated to Prof. Hiroyuki Yamamoto.1 Both authors contributed equally to this work.2To whom correspondence should be addressed. Tel.: 805-893-2817; Fax:

805-893-7998; E-mail: [email protected] abbreviations used are: Dopa, 3,4-dihydroxyphenyl-L-alanine; C-Man-

Trp, C2-[-D-mannopyranosyl]-tryptophan; MALDI, matrix-assisted laserdesorption ionization; TOF, time-of-flight; OHTrp, hydroytryptophan;HPLC, high pressure liquid chromatography.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 35, pp. 23 344 23352, August 28, 2009 2009 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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Mass SpectrometryThe mass of intact Pvfp-1 was deter-mined by matrix-assisted laser desorption and ionization with

time-of-flight (MALDI-TOF) mass spectrometry (Voyager DE,AB Biosystems, Foster City, CA) in the positive ion mode withdelayed extraction. The MALDI matrix was prepared by dis-solving sinapinic acid (10 mg/ml) in 30 vol % acetonitrile. Puri-fied Pvfp-1 was dissolved in this matrix solution to give a final

concentration between 1 and 10 pmol/l. About 1l of samplewas then spotted to the target plate and allowed to dry undervacuum (500 microns). Sample spots were irradiated at 337 nmusing anN

2laser (LSI, Inc., Cambridge, MA) with a pulse width

of 8 ns and a frequency of 5 Hz. Soft ionization generated singly,doubly, and triply protonated ions of the Pvfp-1 protein vari-ants. The singly and doubly protonated ions of bovine serum

albumin were used as molecular mass calibrants at m/z66,430and 33,215, respectively).

Cloning and Sequencing Pvfp-1 cDNATo obtain the partialcDNA sequence of Pvfp-1,total RNA was first isolated from thephenol gland at the tip ofP. viridis foot tissue using an RNaseplant mini kit from Qiagen. A single freshly dissected foot waspulverized under liquid nitrogen using a mortar and pestle,

after which themini kitprotocol wasadopted verbatim. Follow-ing that, the first strand cDNA was synthesized using Super-script II reverse transcriptase at 50 C with an Adapter primer,5-GGCCACGCGTCG ACTAGTACT(T)

16-3 (Invitrogen).

The product of the reverse-transcribed reaction was used as atemplate for subsequent PCRs.

Assuming repetition of the known trypsinized internal pep-tide sequence APPPAX

1TAX

2K (whereXdenotes an unknown

amino acid residues) from a previous study (12), a degenerate

oligonucleotide (sense, 5-AARGCNCCNCCNCCNGC-3,which corresponds to thesequenceKAPPPA) wasdesignedandcombined with an abridged universal amplification primer(antisense, 5-GGCCACGCGTCGACTAGTAC-3; Invitro-

gen) to amplify the 3 end of Pvfp-1 from reverse transcriptasetemplates. The cloned 3 cDNA end provided the C-terminalsequence of Pvfp-1 along with some 3-untranslated region.

Another degenerate oligonucleotide (sense, 5-GCNGTNT-AYCAYCCNCCNTC-3) was designed on the basis of N-ter-

minal sequence of Pvfp-1 (AVYHPPS) and coupled with a gene-specific reverse primer (antisense, 5-GGTTTGCCATATCC-ACCATAACC-3 corresponding to GYGGYGK) cloned aboveto amplify the 5 end cDNA of Pvfp-1.

Once the cDNA sequences encoding the mature Pvfp-1s

were cloned, a GeneRacer kit (Invitrogen) was used to ob-tain the 5 end untranslated re-gion of the cDNA of Pvfp-1 fromfull-length transcripts by 5 rapidamplification to cDNA ends. PCR

was performed with gene-spe-cific primer (antisense, 5-GCTT-TCCATGCAGTCCATGCAGG-TGGATG-3 (corresponding toHPPAWTAWK) coupled with a

GeneRacer 5 sense primer fromInvitrogen.

Generally, PCR was carried out in25 l of 1 Buffer B (Fisher) and 5

pmol of each primer, 5 mol of each

dNTP, 1 l of first strand reaction,and 2.5 units ofTaqDNA polymerase(Fisher) for 35 cycles on a Robocycler(Stratagene). Each cycle consisted of

30s at94 C, 30 sat50 C, and1 min at72 C, witha final extension of15 min.The PCR products were subjected to1% agarose gel electrophoresis, puri-fied, andcloned into a PCRTA vector

(TOPO TA cloning kit; Invitrogen)and transformed into competentTop10 cells (Invitrogen)for amplifi-cation, purification, and sequencing.

FIGURE 1. Thecommonblue mussel M. edulis andgreen mussel P. viridis areshown with attached byssal threads. Distinct consensus decapeptide repeatsequencesareassociatedwiththerepeatdomainsinfp-1footproteinsofthetwospecies.The amino acidDopa(Y*, right panel) is prominentin mefp-1 repeatsbutabsent from Pvfp-1. O denotes trans-4-hydroxyprolines. Residues denoted asX1andX2 are shown by this study to be derived from tryptophan.

FIGURE 2. Complete protein sequence of Pvfp-1 variant 1 deduced from cDNA. The signal peptide isitalicized, and the mature N terminus is indicated by arrows. Three directly sequenced peptide sequences areunderlined: the N terminus(solid line), decapeptide repeats (peptides e andd, dashed line), andcollagen(pep-tide j, dotted line). The single Dopa (Y) residue is circled. The variant 2 sequence is in the supporting data. TheGenBankTM accession numbers for variants 1 and 2 are AAY46226.1 and AAY46227.1.

GreenMussel Adhesive Protein

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Byssal Thread AnalysisFifteen to twenty threads were sev-ered from the proximal end of each byssal stem, washed in 500volumes of MilliQ water, and blotted dry. The dry threads wereweighed and hydrolyzed by one of two methods: 6 M HCl for all

amino acids except for Trp and 4 M NaOH for Trp (15). Acidhydrolysis was done with reusable hydrolysis vials (Pierce) at110 C for 24 h and flash evaporated under high vacuum,whereas NaOH hydrolysates were titrated to pH 34 with gla-cial acetic acid. Purification of Trp-derived amino acids was

achieved by C-18 HPLC (Brownlee Aquapore AOD-300

, 7 m,4.6 250 mm) over an acetonitrile gradient of 010%. Theeluate was monitored continuously at 220 and 280 nm, and allof those peaks absorbing at both wavelengths were collected for

analysis by electrospray ionizationand tandem mass spectrometry.

For routine amino acid analysis,the purified Pvfp-1s were hydro-lyzed in one of three ways: 1) 4 Mmethanesulfonic acid with 0.5%3-methyl-indole (16), 2) 6 M HCl

with 5% phenol in vacuo at 110 Cfor 24 h, or 3) 4 M NaOH in vacuo at110 C for 24 h (15). The firstmethod was dropped because of

extremely low tryptophan deriva-tive recovery. The HCl hydrolysatewas flash evaporated at 50 C undervacuum and shaken to dryness with

0.5 ml of MilliQ water followed

by methanol; NaOH hydrolysateswere titrated to pH 4. Amino acidanalysis was performed accordingto conditions described earlier

with a Beckman System 6300 autoanalyzer (14) on which an auth-entic C2-(-D-mannopyranosyl)-tryptophan (C-ManTrp) standard(17) eluted just after Met with a

run time of 31.5 min.Trp-containing PeptidesPrepa-

ration of tryptic peptides fromPvfp-1,including purification by C-18 HPLC(Brownlee, A

300, 4.6 260 mm) was

done exactly as described by Ohkawaet al. (12). Proteolysis was stopped byacidification to pH 4 with glacial ace-tic acid before HPLC. Three peakfractions corresponding to peptides d,

e, and j described by Ohkawa wereselected for analysis by electrosprayionization mass spectrometry fol-lowed by tandem mass spectrometrywith collision-induced decomposi-

tion using the PE SciexQStar quadru-pole/time-of-flight tandem massspectrometer (PerkinElmer Life Sci-ence) intheUniversity of California at

Santa Barbara Mass Spectrometry

Facility. Interpretation of fragments produced from the peptidesby collision-induced decomposition was assisted by comparisonwith an authentic C-ManTrp standard and by mock fragmenta-tionof various model sequences using Protein Prospector, version

5.1.4, on ExPASy Tools.UV-visible Spectra of Indole DerivativesChemical modifi-

cations based on 1-nitroso-2-naphthylation (18) and nitration(19) were performed as described. The former reacts only withthe 5-hydroxy indole isomer, and the latter, which was devel-

oped for catechol detection, gave a bright yellow color forPvfp-1 and its tryptic peptides. UV-visible spectra wereobtained for 0.1 mM solutions of L-tryptophan (Sigma),C2-mannosyltryptophan (donated by S. Manabe), peptide e,

FIGURE 3. C-18HPLCof C2-hexosylTrp fromNaOH hydrolyzed P. viridis byssal threads (top panel), stand-ard L-Trp (middle panel), and standard C2-mannosyltryptophan (bottom panel). Fractions at 2225 minwere sampled by electrospray ionization mass spectrometry and collision-induced decomposition. Standard7-hydroxytryptophan, which does not survive NaOH hydrolysis, eluted at 17 min.

GreenMusselAdhesive Protein


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and four hydroxyindoles (4-hydroxyindole (Acros Organics),5-hydroxytryptophan (Sigma), 6-hydroxyindole (OakwoodProducts, Columbia, SC), and 7-hydroytryptophan (SynChemOHG, Felsberg, Germany)) using an HP model 8453 UV-visible

scanning spectrophotometer with UV-visible ChemStation(Rev 06.03). All of the solutions were buffered with 5% (v/v)acetic acid and scanned between 235 and 335 nm in masked40-l quartz cells (Starna, Atascadero, CA).

RESULTS

A previous investigation by Ohkawa et al. (12) reported thefollowing attributes for Pvfp-1: 1) an apparent mass of 89 kDabased on SDS-PAGE, 2) marked quinone-like redox cycling

activity without Dopa, 3) significantcarbohydrate content, particularly

with respect to mannose, N-acetyl-glucosamine and fucose, and 4) anEdman-derived primary sequencedominated by two closely relatedconsensus repeats: AOOOAX

1

TAX2K and APOKOX1TAX2K, inwhich O denotes trans-4-hy-droxyproline. TheX

1and/orX

2posi-

tions were consistent with the pres-

ence of an aromatic amino acid butcould not be reconciled with anyknown modification of Tyr or Dopa.

In the present investigation, puri-fied Pvfp-1 was shown to consist of

at least two variants (supplementalFig. S1) with masses of 50 and 64kDa observed by MALDI-TOF massspectrometry (supplemental Fig.

S2). TheN-terminal sequence of theisolated protein was found to be(A)VY*HPPSX

1TAX

2IAOK (sup-

plemental Fig. S1), where X1

and X2

represent Edman cycles devoid

of detectable phenylthiohydantoinderivatives. Y* denotes Dopa andwas the only Dopa detected inPvfp-1.

To further explore the chemistry

of X1

and X2, we first deduced the

complete sequence of Pvfp-1 fromits corresponding cDNA preparedby standard cloning procedures.Two Pvfp-1 variants, probably

related by alternative splicing, werefound with the shorter differingfrom the longer by exactly 15decapeptide repeats. In the longervariant 1 (calculated mass, 61 kDa),

APPPAWTAWK and ATPKP-WTAWK consensus repeats occur14 and 29 times, respectively (Fig. 2and supplemental Fig. S3). Other

intriguing features in Pvfp-1 are a

distinct collagen-like sequence with 18 tripeptide Gly-X-Yrepeats, followed by a short interval (14 amino acids long),before returning to a Trp-rich C-terminal sequence. It musttherefore be concluded that in Pvfp-1 some modification of

tryptophan, not tyrosine, provides the basis for the unknownaromatic amino acids in the decapeptide repeats.

Because tryptophan is unstable to hydrolysis by HCl, puri-fied Pvfp-1andP. viridis byssal threads were hydrolyzed with4 M NaOH. After hydrolysis, component aromatic amino

acids were chromatographically separated by C-18 HPLC(Fig. 3) and analyzed by mass spectrometry to help deducetheir relationship to tryptophan. Two ions with masses of367 and529 Da eluting between22 and 24 min were detected;

FIGURE4.Massspectrometricanalysisof them/z367peak obtainedfrom C-18HPLCof NaOHhydrolyzedP. viridis threads (top panel) and purified Pvfp-1 (bottom panel). The loss of 120 Da is a signature forC-hexosylation.


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the latter decomposed readily to 367 through a loss of 162

Da (hexose) (supplemental Fig. S4), suggesting a cleavage

that is typical of O- and N-linked hexoses (20, 21). The367-Da ion and its fragmentation, most notably the 120-Daloss to m/z 247, are identical with standard C2-[-D-man-nopyranosyl]-tryptophan (22, 23) (Fig. 4). The hexose linked

to Trp in Pvfp-1 is likely to be mannose, but tandem massspectrometry by itself is unable to distinguish different hex-oses. Because C-ManTrp elutes just after Met in amino acidanalysis, this method was used to estimate a C2-mannosyl-tryptophan content of 12dry weight % in byssal threads and

34 dry weight % in Pvfp-1 following base hydrolysis (sup-plemental Table S1). Only trace levels of Trp could bedetected in Pvfp-1; thus the Trp in byssus must come fromother proteins.

To detect modified tryptophansmore directly in the decapeptide

sequences of Pvfp-1, we used tan-dem mass spectrometry with colli-sion-induced decomposition ofthree peptides following trypsindigestion of Pvfp-1 (supplemental

Fig. S5). Peptides d and e wereselected because 75% of theirsequences had already been estab-lished by Edman chemistry (12).

The doubly charged parent ion(M2H)2 (m/z953.8) forthe tryp-tic decapeptide d readily decom-poses to another ion (M2H)2

(m/z 771.25) after a neutral loss of

162 to m/z873 (hexose) followed byanother neutral loss of 204 to m/z771(N-acetyl hexose) (Fig. 5A). Theoxonium ion of hexosyl-N-acetyl-

hexose is evident at m/z 366. The771.25 peak loses water to becomem/z762, which then undergoes the120 Da (60 2) neutral loss typicalof C2-mannosylation; y

6or

OW1TAW

2K (m/z 1144) does the

same. The internal fragment,OW

1TA (m/z 616.1), indicates a

366-Da mass for X1

(Fig. 5B). Nota-bly, the 511.1 (y

2) and 290 (y

3

2-

2H) ions corresponding to W2

KandAW

2K are larger than the compara-

ble W1

-containing fragments by 16Da. C-ManTrp 16 is consistentwith C2-hexosylated hydroxytryp-

tophan (W2K) and the quinoid

counterpart of C2-hexosylatedhydroxytryptophan (AW

2K) (Fig.

5B). A complete annotation of frag-

ment masses is given in supplemen-tal Fig. S6.

Mannose is plausible as theC2-hexose given that WX

1X2W is a

well accepted sequence signature for C2-mannosylation of

tryptophan in proteins (20, 2426). The precise ring assign-

ment of the hydroxyl group in hydroxytryptophan (OHTrp) isnot possible from electrospray ionization tandem mass spec-trometry, but a phenyl ring placement is consistent with theloss of two hydrogens to form a doubly protonated quinoid ion

at m/z290 and by the strong quinone-like redox cycling activityin Pvfp-1 and tryptic decapeptides (12).

Peptide e (m/z757) is less glycosylated than peptide d butshowssome trends similar to those in the 771 ion during fragmentation(Fig.6).They

8ion(OPAW

1TAW

2K)at m/z1328 showsa neutral

loss of 120 Da. The fragment ions implicate both W1

and W2

asC2-[-D-mannopyranosyl]-hydroxytryptophan; the y

2(W

2K)

and y4

ions (TAW2K) are particularly suggestive of W

2(Fig. 6;

complete fragment annotation in supplemental Fig. S7).

FIGURE 5. Tryptic peptide d (parent ions [M 2H]2 m/z953and 771)following collision-induceddecom-position. O, trans-4-hydroxyproline; W

1, C2-hexosylTrp; W

2, C2-hexosylhydroxyTrp. See supporting data for

complete fragment annotation.


http://www.jbc.org/cgi/content/full/M109.022517/DC1http://www.jbc.org/cgi/content/full/M109.022517/DC1http://www.jbc.org/cgi/content/full/M109.022517/DC1http://www.jbc.org/cgi/content/full/M109.022517/DC1http://www.jbc.org/cgi/content/full/M109.022517/DC1http://www.jbc.org/cgi/content/full/M109.022517/DC1http://www.jbc.org/cgi/content/full/M109.022517/DC1http://www.jbc.org/cgi/content/full/M109.022517/DC1http://www.jbc.org/cgi/content/full/M109.022517/DC1http://www.jbc.org/cgi/content/full/M109.022517/DC1http://www.jbc.org/cgi/content/full/M109.022517/DC1http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/cgi/content/full/M109.022517/DC1http://www.jbc.org/cgi/content/full/M109.022517/DC1http://www.jbc.org/cgi/content/full/M109.022517/DC1http://www.jbc.org/cgi/content/full/M109.022517/DC1http://www.jbc.org/cgi/content/full/M109.022517/DC1http://www.jbc.org/cgi/content/full/M109.022517/DC1http://www.jbc.org/cgi/content/full/M109.022517/DC1http://www.jbc.org/cgi/content/full/M109.022517/DC1

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The fragmentation of peptide j (Fig. 7 and supplemental Fig.S8) was undertaken to corroborate the odd collagen-like pri-mary structure predicted by the cDNA-deduced Pvfp-1

sequence (Fig. 2). Peptide j is 14 residues long, and itssequence corresponds to residues 496 509 with Gly at everythird residue and three of the five prolines converted totrans-4-hydroxyproline.

Neither hydroxytryptophan nor its C-hexosylated forms

were detected after methansulfonic acid or NaOH hydrolysis ofthe byssus or Pvfp-1. We observed that whereas standard 5- or7-hydroxytryptophan were stable to hydrolysis in methansulfo-nic acid, standard C-ManTrp was not. OHTrp standards

hydrolyzed in the presence of sugarswere not detected by amino acid

composition. C2-[-D-mannopyr-anosyl]-hydroxytryptophan wouldthus be unlikely to survive meth-ansulfonic acid. OHTrp recoveryfollowing alkaline hydrolysis is pre-

vented by the formation of quinoni-mine at alkaline pH (27).Tandem mass spectrometry sup-

ports the presence of OHTrp but is

unable to specify the position of thehydroxy group. To further clarifythis important point, Pvfp-1 andpeptide e were reacted withnitrosonaphthol, which is specific

for the 5-hydroxy position on theindole (18); both were negative. Inaddition, the UV absorbance spec-trum of peptide e at pH 3.5 was

compared with a series of relatedindole derivatives (28, 29). In con-trast to L-tryptophan and C-Man-Trp, peptide e has a

maxat 269 nm

shifted to a lower wavelength (Fig. 8

and supplemental Fig. S9), and anextinction coefficient of 18,000 M1

cm1 at 269 nm that is consistentwith the presence of two modifiedtryptophans enhanced by mannosy-

lation (28). Of the four hydroxy-in-dole derivatives tested, only 7-hy-droxytryptophan shared the samemax

values as peptide e. Indeed, thetwo spectra are nearly superimpos-

able between 250 and 300 nm.Although future studies need to fur-ther characterize the tryptophanchemistry of peptide e by protonNMR and electrochemistry, the

identification W2

as C2 hexosyl-7-hydroxy tryptophan in Pvfp-1appears reasonable. A final test ofhydroxy-indole reactivity with

Arnows reagent showed that only

7-hydroxy-tryptophan producedthe same bright yellow product formed by Pvfp-1 (supplemen-tal Fig. S9).

DISCUSSION

Pvfp-1 resembles other mussel coating proteins in its canon-ical, Pro- and Lys-rich repeats. It diverges significantly, how-ever, in its reliance on modifications of tryptophan rather thantyrosine for its redox chemistry. Pvfp-1 consists of two closely

related variants, the larger of which has predicted and observedmasses of 61 and 64 kDa, respectively. Both variants show ahighly repetitive sequence with four distinct domains: a Trp-rich decapeptide repeat (consensus: APPPAWTAWK and

FIGURE 6. Tryptic peptide e (parent ion[M 2H]2 m/z757) following collision-induced decomposition.O denotes trans-4-hydroxyproline; W1 andW2 both areconsistentwith C

2-hexosylhydroxyTrp. See supportingdata for complete fragment annotation.

FIGURE 7. Tryptic peptide j (parent ion [M 2H]2 m/z 692) after collision-induced decomposition.O denotes trans-4-hydroxyproline. See supporting data for complete fragment annotation.


http://www.jbc.org/cgi/content/full/M109.022517/DC1http://www.jbc.org/cgi/content/full/M109.022517/DC1http://www.jbc.org/cgi/content/full/M109.022517/DC1http://www.jbc.org/cgi/content/full/M109.022517/DC1http://www.jbc.org/cgi/content/full/M109.022517/DC1http://www.jbc.org/cgi/content/full/M109.022517/DC1http://www.jbc.org/cgi/content/full/M109.022517/DC1http://www.jbc.org/cgi/content/full/M109.022517/DC1http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/cgi/content/full/M109.022517/DC1http://www.jbc.org/cgi/content/full/M109.022517/DC1http://www.jbc.org/cgi/content/full/M109.022517/DC1http://www.jbc.org/cgi/content/full/M109.022517/DC1http://www.jbc.org/cgi/content/full/M109.022517/DC1

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ATPKPWTAWK) domain largely in the N-terminal two-thirds, a collagen domain (Gly-X-Y repeats), a short hinge (res-

idues 450 459), and a predicted, short coiled-coil regionbefore returning to Trp-rich repeats at the C terminus (Fig. 9).The collagen domain is reminiscent of other collagen-like pro-teins such as the macrophage scavenger receptor (30), Torpedo

acetylcholinesterase (31), and com-plement C1q (32), in which a small

collagen domain directs trimer for-mation. The proposed trimericstructure remains speculative forPvfp-1 because only the single chainmass has been determined with any

accuracy by MALDI-TOF.Pvfp-1 exhibits extensive post-translational decoration. Pro is tar-geted for hydroxylation throughout

both decapeptide consensus repeatsand in the collagen domains; Thr-2in ATPKPWTAWK appears to beO-glycosylated with O-(N-acetyl)-hexosyl-hexose, which agrees with

the high levels ofN-acetyl-glucosa-mine reported earlier (12) but dif-fers from the previous designationof Pro for position 2 in peptide e.

Possibly, the phenylthiohydantoinderivative of glycosylated Thr hasthe same elution time as Pro-phe-nylthiohydantoin on C-18 HPLC.Notably, Trp undergoes C2-hexosy-

lation (probably by mannose) andhydroxylation. Whereas W

1andW

2

are always hexosylated, W2

seemsfavored for hydroxylation. Peptidesd and e may be representative of

other decapeptides in Pvfp-1 withrespect to C-hexosylation andhydroxylation. At this stage, how-ever, aside from ample evidence for120-Da mass losses, i.e. C-hexosyl-

Trp in other peptides, collision-in-duced decomposition of many pep-tides remains too complex forconfident interpretation. Pvfp-1 hasonly a single Dopa located near the

N terminus.C-linked mannosylation of pro-

teins is an unusual but not unprece-dented modification of tryptophan.

It was first reported in pancreatic

ribonuclease and since then in over47 other proteins including notablythrombospondin and complementproteins with the sequence motif

WX1X2

W (20,2226). Although thefunction of this modificationremains elusive, mannosylation is

known to render the tryptophan more polar and solvent-acces-sible (23). In complement proteins, C-ManTrp in the throm-

bospondin-like repeat domains has been proposed to have anadhesive function (33). The potential presence of up to 80C-ManTrp residues in Pvfp-1, which functions as an adhesiveand coating in byssus (911), begs the hypothesis that manno-

FIGURE 8. Ultraviolet absorbance spectra of Pvfp-1-derived peptide e and model hydroxyindolesin 5%acetic acidwith extinction coefficientsat selectedwavelengths. Top panel, 4-hydroxyindole. Second panel,5-hydroxytryptophan. Third panel, 6-hydroxyindole. Bottom panel, 7-hydroxytryptophan. The spectrum ofpeptide e (broken line) is superimposed with all other spectra.



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sylation makes tryptophan behave more like Dopa: sticky and

prone to cross-link formation. This conjecture, however, is toosimple because tryptophan needs to be hydroxylated before

becoming redox active like Dopa. 5-Hydroxytryptophan in

engineered proteins and as a free amino acid was reported to

undergo oxidation resulting in the formation ofp-quinonimineand di-Trp cross-links (27, 34). Indeed, based on these studies,

we expected to find C2-mannosylated 5-HOTrp in peptide e

and Pvfp-1. UV spectra, however, support the rarer 7-OHrather than the 5-OH isomer and represent the first report of

naturally occurring 7-hydroxytryptophan in proteins (35).

7-OHTrp seems a better mimic of Dopa than the other isomers.

Upon oxidation, it forms an o-quinonimine (Fig. 10A), and theindolic N-H and phenolic OH groups are close enough to che-

late metal ions (Fig. 10A), a common capability of musselbyssus

(9, 36).

If 7-hydroxylation makes Trp more like Dopa, why botherwith mannosylation? Trp mannosylation was observed to block

cleavage in the vicinity of modified residues by exo- and endo-

proteases, hence rendering proteins more resistant to degrada-tion (28). In addition, Trp-rich proteins are quite hydrophobic

and thus prone to aggregation in solution (37). C-mannosyla-

tion of Trpresidues appears to make them fully water accessible

while preventing aggregation (38).

In summary, the green mussel P. viridis, employs an intrigu-ing alternative to Dopa in Pvfp-1, one of its byssal adhesive

proteins. Although much bulkier, C-Man-7-OHTrp resemblesDopa in having attributes that contribute to both cohesive and

adsorptive interactions necessary for adhesion (39).

With respect to cohesion, the two-electron oxidation ofC-Man-7-OHTrp to an o-quinonimine (Fig. 10A) mimics

Dopa-o-quinone, which is known to form covalent cross-links

with other Dopa, cysteine, lysine, and histidine residues (Fig.10B) (7, 40). The coordination of metal ions via the o-hydroxyl

groups in Dopa also contributes to cohesion, particularly in the

byssal cuticle ofMytilus galloprovincialis (9). The ortho-place-ment of indolic-N and phenolic OH groups in C-Man-7-

OHTrp seems well suitedto metal ion chelation, but this hasyet

to be investigated.The stickiness of adhesive molecules is determined by their

adsorptive tendencies. Dopa complexation of metal hydroxideson surfaces provides strong and reversible interactions (Fig.10B) (41). Again, given the ortho-placement of electronegative

elements, such interactions also seem likely with C-Man-7-OHTrp. Future studies need to closely examine what adaptiveadvantages C-Man-7-OHTrp offers over Dopa.

AcknowledgmentsWe thank S. Manabe and Y. Ito (RIKEN) for syn-

thetic C2-(-D-mannopyranosyl)-tryptophan and K. Ohkawa (Shin-

shu University) for numerous discussions. C. Ross and M. Gilg (Uni-

versity of North Florida) and A. Benson and M. Blouin (U.S.

Geological Survey) generously provided P. viridis from Tampa, FL.

FIGURE 9. Model of trimeric Pvfp-1 based on the trimerization imposedby formationof thecollagendomain. Structure of thelong N-terminal andshortC-terminal repeat domains is not predictable at present. The coiled-coilregion was predicted by Coils (window 14) in ExPAsy Tools.

FIGURE 10. C2-hexosyl-7-OHTrp reactivity (A) has parallelswith peptidyl-Dopa (B). In the redox pathway, C2-hexosyl-OHTrp loses two hydrogens tobecome an o-quinonimine (bottom); Dopa loses twohydrogens (2H 2e)to becomean o-quinonethat reacts with cysteine, histidine, lysine, and otherDopa residues to form cross-links (B). In the metal chelate pathway, both theindolic nitrogen andphenolic oxygenof C2-hexosyl-7-OHTrpshould contrib-ute electrons for a bidentate complexation (A, top right); Dopa forms stablebidentate complexes with metal ions and metal hydroxides (Mn[OH]

n)

(B, center). Thepreferred metal (M) bound to Dopain Mytilus byssusis FeIII; inPvfp-1 ofP. viridis byssus it is unknown.Covalent cross-links andbis-bidentatemetalcomplexes contribute to adhesive cohesion,whereas mono-bidentatemetal hydroxide complexes contribute to adhesive adsorption.



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